Frequently, it is desirable to radiolabel bioactive molecules with radioactive iodine or other halogens such as bromine. Most commonly, radioactive iodine is supplied in the form of KI* (wherein * represents a radioisotope of the halogen of interest, e.g., I.sup.125 or I.sup.131), and the labeling procedure involves reaction of KI* with chloramine-T (CAT) to release I.sub.2 * followed by addition of the substrate which then takes up the I.sub.2 * to form a radioiodinated product.
A major drawback to this procedure occurs when the substrate (compound to be labeled) is sensitive to oxidation or chlorination. Poor yields of the desired radiohalogenated or radioiodinated product and possibly complete decomposition of the substrate may occur through oxidation or chlorination, if residual CAT is left in the solution when the substrate is added.
The IODOBEADS.RTM. system (Pearce Chemical Company) attempts to avoid the problem of substrate decomposition by bonding the CAT onto polymer beads. A KI* solution is added to the beads and shaken to release free I*.sub.2. A solution of the substrate is then added and shaken until the brown color disappears, then the beads are immediately filtered off to remove any residual CAT. The radioiodinated product is separated from the solution by preparative HPLC.
This system is only partially successful because CAT is released from the beads very rapidly. With sensitive substrates, if the manipulations are not carried out very quickly, significant amounts of CAT will appear in the solution and decompose the substrate. In many cases, the yields are very poor even though the procedure is carried out as quickly as possible. Finally, preparation of the polymer beads is relatively expensive.
Methods are known for radioiodinating molecules using chloramine-T (sodium-p-toluenesulfochloramine).
U.S. Pat. No. 3,867,518 to Coffey et al. describes a radioimmunoassay for insulin and further describes a method for the preparation of radioiodinated insulin using chloramine-T (sodium-p-toluenesulfochloramine) and a phosphate buffer. Coffey et al. does not disclose or suggest the use of piperidine or N-chloro-piperidine in a method of radioiodination.
U.S. Pat. No. 4,196,185 to Focella discloses an immunoassay for phencyclidine. To prepare radioiodinated N-(4-hydroxy-2-phenelthyl)-4-[1-piperidinyl)cyclohexyl] benzene acid sulfate, the compound was placed in ethyl acetate and water was added to a vial containing Na.sup.125 I. To this mixture was added chloramine-T. Then sodium meta bisulfite was added to stop the reaction.
U.S. Pat. No. 4,436,718 to Smith discloses an iodinating reagent. This patent discloses that N-chloro-4-methylbenzenesulfonamide or chloramine-T has long been used as the oxidant to mediate iodination reactions. The disclosure states that the use of chloramine-T has its drawbacks. The oxidant of Smith comprises a water insoluble bead having covalently attached to its surface molecules a chloramine group, with a size and shape of the bead being compatible with easy physical separation of the oxidant from a solution of the biological specimen. In the invention of Smith, chlorosulfonic acid, aqueous ammonia, and sodium hypochlorite yield the most preferred N-chlorobenzene sulfonamide functionality on the beads.
U.S. Pat. No. 4,450,149 to Kabalka et al. discloses a radiohalogenation method. In the method of Kabalka, the general procedure for iodination involved adding methanol and organoborane. Iodine-mono-chloride is then added. Sodium thiosulfate is then added to the mixture and the layers are dried over magnesium sulfite. Benzoyl chloride was added to 4-penten-1-ol in pyridine and stirred overnight at room temperature. Sequentially, diluted HCl was added and the mixture was saturated with aqueous Na.sub.2 CO.sub.3 and water.
U.S. Pat. No. 4,528,134 to Stentz et al. discloses a method of iodinating insulin using chloramine-T. U.S. Pat. No. 4,591,552 to Neurath discloses the detection of hepatitis B surface antigen with labelled synthetic peptide. This patent discloses that if a peptide contains tyrosine it can be labelled by contacting the peptide with sodium I.sup.125 in a buffer and then oxidizing it in the presence of chloramine-T. Neurath discloses that the peptide can be labelled with or without a solid support such as polystyrene beads. U.S. Pat. No. 4,775,638 to Haisma discloses a single vial technique for radiolabelling protein. Haisma discloses use of coupling agents such as iodogen (1,3,4,6-tetrachloro-3,6-diphenylglycuril), chloramine-T, lactoperoxidase and iodine monochloride. Iodination is conducted on an ion exchange resin.
U.S. Pat. No. 4,874,601 to Flanagan discloses a radiolabelling kit. The radiolabelling kit of Flanagan includes oxidizing agents such the sodium salt of N-chloro-p-toluenesolufonamide or chloramine-T, iodogen, or iodobeads (chloramine-T bonded to polystyrene beads). Flanagan does not disclose or suggest the use of piperidine or N-chloro-piperidine in a method of radioiodination.
U.S. Pat. No. 4,966,999 to Coughlin et al. discloses radiohalogenated compounds for site specific labelling. The process of Coughlin et al. includes an oxidant such as chloramine-T, iodobeads or iodogen. Coughlin does not disclose or suggest the use of piperidine or N-chloro-piperidine in a method of radioiodination.
U.S. Pat. No. 4,994,258 to Burns et al. discloses novel radiolabelled antagonists for pancreatic imaging. The radiolabelling process takes place in a sodium hydroxide solution of Na.sup.125 I in the presence of cupric sulfate and ammonium sulfate.
U.S. Pat. No. 5,084,266 to McKenzie et al. discloses a method for tumor imaging utilizing a labelled tumor specific antibody and a non-tumor reactive antibody. McKenzie et al. found that the chloramine-T method for radiolabelling resulted in a greater loss of immunoreactivity than either the iodobead or enzymobead method. The enzymobead method was the method of choice for labelling in the method of McKenzie et al.
U.S. Pat. No. 5,171,666 to Gutowski et al. discloses the labelling of immunoglobulins using chloramine-T.
The publication of Markussen, J. et al., Insulin: Chem., Struc. Funct. Insulin Relat. Horm., Proc. Int., Insulin Symp. (1980) pp. 161-168 is entitled "The Application of HPLC to the Analysis of Radioiodinated Tracers of Glucagon and Insulin". This publication discloses the use of chloramine T and lactoperoxidase in the radioiodination of glucagon.
The publication of Boothe, Thomas E. et al., J. Labelled Compd. Radiopharm., Vol. 23, (1986) pp. 479-485, entitled "The Preparation of No-carrier Added 4-[.sup.131 I] iodoantipyrine using chloramine-T. The publication discloses the use of chloramine-T and iodogen to iodinate antipyrine for use as a tracer in studying cerebral blood flow.
Dickinson, Kenneth E., et al., Mol. Pharmacol., Vol. 26, No. 2, (1984) pp. 187-195. "Photoaffinity Crosslinking of a Radioiodinated Probe .sup.125 I-A55453, into Alpha 1--Adrenergic Receptors" discloses the radioiodination of an alpha1-adrenergic receptor probe. The method of radioiodination includes the use of chloramine-T and Na.sup.125 I.
Youfeng, He, et al., J. Labelled Compd. Radiopharm., Vol. 19, (1982) pp. 807-819, "A Comparative Study of Radioiodination of Simple Aromatic Compounds via N-Halo-Succinimides and Chloramine-T in Trifluoroacetic Anhydride (TFAA)". This paper reports the comparative results of radioiodination performed using N-chlorotetrafluorosuccinimide, N-chlorosuccinimide, N-bromosuccinimide, and chloramine-T (CAT) as oxidizing agents with TFAA as an aprotic solvent. The study concluded that CAT is a superior oxidizing agent for the iodination of simple benzenes which are water insoluble but which are soluble in TFAA.
Nakamura, Masahiro, et al., Radioisoptopes, Vol. 40, No. 3, (1991) pp. 112-117, "Preparation and Evaluation of Radio Labelled Recombinant Human Interleukin-2 by Improved Chloramine-T Method". This publication discloses the use of a very small quantity of chloramine T to activate iodine (21 fold excess to iodine in molar ratio). This method of iododination is useful for iodination of unstable peptides and proteins.
Baindur, Nandkishore, et al., J. Med. Chem., Vol. 31, No. 11, (1988) pp. 2069-2071, "Discloses a Photoaffinity Label for the D-1 Dopamine Receptor". Radioiodination methodology used by Baidur includes the use of a modified NaI-Chloramine-T procedure. None of the above patents or publications disclose or suggest the use of piperidine or N-chloro-piperidine in a method of radioiodination.
The method of radiohalogenation of the present invention overcomes the deficiencies of prior art methods and provides an inexpensive method for radiohalogenating biomolecules which is non-destructive to the biomolecule .